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Cancer Detection and Prognosis
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Santiago Ropero, Manel Esteller
Several molecular methods have been developed for detecting global changes in DNA methylation in CpG islands, including HPLC, methyl-acceptance assay, differential methylation hybridization, Ms-arbitrarily primed PCR, and RLGS. The use of RLGS for analyzing global CpG island hyper-methylation requires methylation-sensitive restriction enzymes whose restriction sites are preferentially located in CpG islands. Two restriction enzymes are currently used: NotI and AscI. The two-dimensional profiles of landmark sites of normal tissues and tumor samples are compared, allowing the identification of DNA sequences that are differentially methylated in tumor samples (Figure 14.1).
Lacrimal Gland as a Target Organ for Adenovirus Gene Therapy Encoding Erythropoietin for Dry Eye Induced by Benzalkonium Chloride
Published in Current Eye Research, 2021
Lara Cristina Dias, Changyu Zheng, Adriana de Andrade Batista Murashima, Ana Carolina Dias, Marina Zilio Fantucci, Luiz Fernando Nominato, Lilian Eslaine Costa Mendes da Silva, Eduardo Melani Rocha
RLGs from the three groups (control and Ad-hEpo LG) were homogenized in a solution containing 10 mM Tris Buffer, pH 7.4, 150 mM, 1% Triton X100 and Complete Protease Inhibitor cocktail tablet (Roche Diagnostic Co, Indianapolis, IN, USA) prepared as indicated by the provider, with an Omni TH homogenizer (Omni International, Kennesaw GA, USA) and stored at −20°C until ELISA evaluation.
Emerging DNA methylation inhibitors for cancer therapy: challenges and prospects
Published in Expert Review of Precision Medicine and Drug Development, 2019
Aurora Gonzalez-Fierro, Alfonso Dueñas-González
There are numerous reports demonstrating that tumor suppressor genes belonging to nearly every cancer pathway or function category have silenced or diminished expression due to abnormal promoter hypermethylation. Many of these reports are summarized in reviews [72–74]. In fact, these findings were the rationale behind the clinical development of DNA methyltransferase inhibitors. The first discoveries of promoter methylated tumor suppressor genes were made using the candidate gene approach. Greger et al., and Sakai et al. [75,76], were the first to demonstrate silencing of retinoblastoma gene by promoter methylation in primary tumors as a ‘non-genetic’ structural defect mechanism of allele inactivation according to the ‘two-hit’ Knudson hypothesis [77]. In 2001, Esteller et al. performed a comprehensive analysis of promoter hypermethylation changes in 12 genes (p16, p14ARF, p15, p73, APC, BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK). Each of these genes was rigorously characterized for association with abnormal gene silencing from over 600 primary tumor samples representing 15 major tumor types. They found a unique profile of promoter hypermethylation for each human cancer in which some gene changes are shared and others are cancer-type specific. These data illustrated that epigenetic inactivation may affect genes implicated in key cancer molecular pathways such as cell immortalization and transformation cycle, DNA repair, cell adherence, metastases, and metabolic enzymes among others [74]. The main findings of this study were also somehow replicated by Costello et al., using a ‘non-candidate approach’. They performed a global analysis of the methylation status of 1,184 unselected CpG islands in 98 primary human tumors using restriction landmark genomic scanning. They reported an average of 600 CpG islands found methylated in tumors, and again, methylation patterns that were shared within each tumor type, together with patterns and targets that displayed distinct tumor-type specificity. The functional consequences of such methylation were inferred from treating glioma cell lines with known methylated and silenced genes with decitabine. Overall, 6 out of 16 genes (38%) were fully or partially reactivated while 10 genes (63%) were unaffected by the demethylation treatment in all five cell lines, regardless of methylation status [78].
Evolving paradigms for the biological response to low dose ionizing radiation; the role of epigenetics
Published in International Journal of Radiation Biology, 2018
Paul N. Schofield, Monika Kondratowicz
At this point, it is worth considering how the development of technology for studying DNA methylation has impacted on the development of the epigenetic paradigm in IR action. Much of the early work carried out on DNA methylation in irradiated cells or organisms was dependent for its resolution on the techniques available at the time (Harrison and Parle-McDermott 2011). For example, before 1990, HPLC-dependent methods were the main methodology used to look at overall global changes in 5meC status across the whole genome and only methylation sensitive isoschizomer methylation-sensitive restriction enzyme methods were available which depended on knowledge of the sequence involved and an appropriate PCR being available. The development of anti-5meC antibodies extended the ability to detect methylated regions of the genome through immunoprecipitation (Me-DIP), but the most important developments in terms of screening for specific differentially methylated regions were of random primed methylation sensitive PCR and restriction landmark genomic scanning and related techniques. These were limited by lack of sensitivity resulting in skewing of results towards repetitive elements but nevertheless permitted discovery of unknown differentially methylated loci. It was only with advances in detection and quantification of DNA modifications using sequencing methodologies such as pyrosequencing and next generation sequencing that specific epigenetic modification could be characterized at scale. The main methods currently used include bisulphite extension (methylation changes to a single base can be detected within the region of interest or else averaged within a locus), DNA Extension Assays; MSP-PCR; High-Resolution Melt (HRM); and microarray or bead array (Infinium, EPIC850 chips) (Kurdyukov and Bullock 2016). The most recent developments in DNA methylation sequencing include Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al. 2005) and more recently the bead chip method using single base extension on bisulphite converted DNA and hybridization to more than 850,000 oligonucleotides (Pidsley et al. 2016). Though this technique is of the highest precision and resolution, it has not yet been used in the study of low dose responses. It is however very clear that our concept of the relationship between NTEs, epigenetics, and methylation marks has been substantially determined by the developments in the technology of methyl DNA determination (Figure 1).